RNA interference screen for human genes associated with West Nile virus infection

Abstract

West Nile virus (WNV), and related flaviviruses such as tick-borne encephalitis, Japanese encephalitis, yellow fever and dengue viruses, constitute a significant global human health problem. However, our understanding of the molecular interaction of such flaviviruses with mammalian host cells is limited. WNV encodes only 10 proteins, implying that it may use many cellular proteins for infection. WNV enters the cytoplasm through pH-dependent endocytosis, undergoes cycles of translation and replication, assembles progeny virions in association with endoplasmic reticulum, and exits along the secretory pathway. RNA interference (RNAi) presents a powerful forward genetics approach to dissect virus-host cell interactions. Here we report the identification of 305 host proteins that affect WNV infection, using a human-genome-wide RNAi screen. Functional clustering of the genes revealed a complex dependence of this virus on host cell physiology, requiring a wide variety of molecules and cellular pathways for successful infection. We further demonstrate a requirement for the ubiquitin ligase CBLL1 in WNV internalization, a post-entry role for the endoplasmic-reticulum-associated degradation pathway in viral infection, and the monocarboxylic acid transporter MCT4 as a viral replication resistance factor. By extending this study to dengue virus, we show that flaviviruses have both overlapping and unique interaction strategies with host cells. This study provides a comprehensive molecular portrait of WNV-human cell interactions that forms a model for understanding single plus-stranded RNA virus infection, and reveals potential antiviral targets.

Figure 1. RNAi screen and bioinformatics

(a) West Nile virus RNAi screen strategy (see text for description). (b–c) Bioinformatics classification of hits into (b) biological process and (c) molecular function categories. * Denotes categories found enriched (p<0.05) relative to all the genes examined in the RNAi screen. Only categories with ten or more members are displayed.

Figure 2. CBLL1 (a–e) and ERAD (f–g) silencing reduces West Nile virus (WNV) infection

(a and b) CBLL1 silencing reduces WNV immunostained cells (MOI ~0.3, 10x, Zeiss). Red (virus), blue (nucleus). (c) a time course analysis showing reduced internalization of TRITC-WNV (MOI ~100) into CBLL1 silenced cells. (d) Localization of TRITC-WNV within CBLL1 silenced cells (percentage of total cells showing the phenotypes, 20 images, ~6 cells each). Plasma membrane alone (PM only), inside the cell (In only), and both PM and inside (In+PM). AP3S2 and vATPase were controls (see text). (e) MG132 reduces WNV internalization (MOI ~100). (f) ERAD network. Yellow squares denote hits; blue circles represent other host proteins within the network neighborhood. Dotted line indicates complexes. (g) ERAD silencing reduces WNV infection (MOI ~0.3). DERL2R is RNAi resistant mutant of DERL2. For (a) and (g), the % of infected control NT-siRNA cells (~30%) was set at 100% and used to normalize the % infection of siRNA-treated cells (from six fields of ~8000 cells). Values for (c) and (e) are number of virus particles/cell (mean of 20 cells). Results are mean ± SD from a representative experiment performed in triplicates.

Figure 3. MCT4 silencing enhances WNV replication

(a and b) MCT4 silencing increases the number of WNV immunostained cells (MOI ~0.3, 10x, Zeiss). Red (virus), blue (nucleus). (c) QPCR of WNV RNA levels in control NT-siRNA and MCT4 silenced cells (ng viral RNA/beta actin). (d) immunostaining for WNV E-protein in MCT4 silenced cells. (e) WNV secretion from MCT4 silenced cells, expressed as plaque forming units/ml (PFU/ml). Values in (a) and (d) are percentage of total cells immunostained for WNV (six images having ~1000 cells each). Results are mean ± SD from a representative experiment performed in triplicates.

Figure 4. (a–c) Interaction of West Nile virus (WNV) and dengue virus (DENV) with host cells

(a) Classification of HSFs common to both WNV and DENV or specific to WNV, into biological process categories. * Denotes categories found enriched (p<0.05) relative to all identified HSFs. (b) Focal adhesion complex (FAC) network. Cyan line connects the core proteins. Yellow squares (WNV specific HSFs), red squares (WNV and DENV shared HSFs), blue circles (other host proteins within the network neighborhood). (c) Effect of silencing of PXN, SHC1 and PITPNM2 on WNV and DENV infection (the % of infected control NT-siRNA cells (~30%) was set at 100% and used to normalize the % infection of siRNA-treated cells (from six fields of ~8000 cells). PITPNM2R indicates RNAi resistant mutant of PITPNM2. Results are mean ± SD from a representative experiment performed in triplicates.